C-terminal Group Research Articles

Influence of substituents on conformational preferences of helix foldamers of γ‐dipeptides

Conformational preferences of 9- and 14-helix foldamers have been studied for γ-dipeptides of 2-aminocyclohexylacetic acid (γAc6 a) residues such as Ac-(γAc6 a)2 -NHMe (1), Ac-(C(α) -Et-γAc6 a)2 -NHMe (2), Ac-(γAc6 a)2 -NHBn (3), and Ac-(C(α) -Et-γAc6 a)2 -NHBn (4) at the M06-2X/cc-pVTZ//M06-2X/6-31 + G(d) level of theory to explore the influence of substituents on their conformational preferences. In the gas phase, the 9-helix foldamer H9 and 14-helix foldamer H14-z are found to be most preferred for dipeptides 2 and 4, respectively, as for dipeptides 1 and 3, which indicates no remarkable influence of the C(α) -ethyl substitution on conformational preferences. The benzyl substitution at the C-terminal end lead H14-z to be the most preferred conformer for dipeptides 3 and 4, whereas it is H9 for dipeptides 1 and 2, which can be ascribed to the favored CH···π interactions between the cyclohexyl group of the first residue and the C-terminal benzyl group. There are only marginal changes in backbone structures and the distances and angles of H-bonds for all local minima by C(α) -ethyl and/or benzyl substitutions. Although vibrational frequencies and intensities of the dipeptide 4 calculated at both M06-2X/6-31 + G(d) and M05-2X/6-31 + G(d) levels of theory are consistent with observed results in the gas phase, H14-z is predicted to be most preferred by ΔG only at the former level of theory. Hydration did not bring the significant changes in backbone structures of helix foldamers for both dipeptide 1 and 4. It is expected that the different substitutions at the C-terminal end lead to the different helix foldamers, which may increase the resistance of helical structures to proteolysis and provide the more surface to the helical structures suitable for molecular recognition.

Roles of Intramolecular and Intermolecular Hydrogen Bonding in a Three-Water-Assisted Mechanism of Succinimide Formation from Aspartic Acid Residues

Aspartic acid (Asp) residues in peptides and proteins are prone to isomerization to the β-form and racemization via a five-membered succinimide intermediate. These nonenzymatic reactions have relevance to aging and age-related diseases. In this paper, we report a three water molecule-assisted, six-step mechanism for the formation of succinimide from Asp residues found by density functional theory calculations. The first two steps constitute a stepwise iminolization of the C-terminal amide group. This iminolization involves a quintuple proton transfer along intramolecular and intermolecular hydrogen bonds formed by the C-terminal amide group, the side-chain carboxyl group, and the three water molecules. After a conformational change (which breaks the intramolecular hydrogen bond involving the iminol nitrogen) and a reorganization of water molecules, the iminol nitrogen nucleophilically attacks the carboxyl carbon of the Asp side chain to form a five-membered ring. This cyclization is accompanied by a triple proton transfer involving two water molecules, so that a gem-diol tetrahedral intermediate is formed. The last step is dehydration of the gem-diol group catalyzed by one water molecule, and this is the rate-determining step. The calculated overall activation barrier (26.7 kcal mol−1) agrees well with an experimental activation energy.

Temperature dependence of C-terminal carboxylic group IR absorptions in the amide I′ region

Studies of structural changes in peptides and proteins using IR spectroscopy often rely on subtle changes in the amide I′ band as a function of temperature. However, these changes can be obscured by the overlap with other absorptions, namely the side-chain and terminal carboxylic groups. The former were the subject of our previous report (Anderson et al., 2014). In this paper we investigate the IR spectra of the asymmetric stretch of α-carboxylic groups for amino acids representing all major types (Gly, Ala, Val, Leu, Ser, Thr, Asp, Glu, Lys, Asn, His, Trp, Pro) as well as the C-terminal groups of three dipeptides (Gly–Gly, Gly–Ala, Ala–Gly) in D2O at neutral pH. Experimental temperature dependent IR spectra were analyzed by fitting of both symmetric and asymmetric pseudo-Voigt functions. Qualitatively the spectra exhibit shifts to higher frequency, loss in intensity and narrowing with increased temperature, similar to that observed previously for the side-chain carboxylic groups of Asp. The observed dependence of the band parameters (frequency, intensity, width and shape) on temperature is in all cases linear: simple linear regression is therefore used to describe the spectral changes. The spectral parameters vary between individual amino acids and show systematic differences between the free amino acids and dipeptides, particularly in the absolute peak frequencies, but the temperature variations are comparable. The relative variations between the dipeptide spectral parameters are most sensitive to the C-terminal amino acid, and follow the trends observed in the free amino acid spectra. General rules for modeling the α-carboxylic IR absorption bands in peptides and proteins as the function of temperature are proposed.

Octaproline, a conformationally flexible chiral selector in liquid chromatographic enantioseparation

A proline octapeptide-derived chiral selector (CS) end-capped using a pivaloyl group was covalently linked to a silica gel chromatographic matrix by the C-terminal group. The chromatographic behaviour of the resulting chiral stationary phase (CSP) using different conditions was compared to those containing 3,5-dimethylphenylcarbamate residues on the proline units. An enantioseparation ability highly dependent on the mobile phase used is observed for these CSPs. When mixtures of alkane/alcohol or alkane/ether are used as mobile phase a similar enantioselectivity is obtained. Nevertheless, in the presence of chlorinated solvents, and without a hydrogen bonding donor in the mobile phase, enantioselectivity is extremely reduced. The reversibility of this phenomenon, attributed to a conformational change in the CS, is examined.

Expression, purification, and biological activity of the recombinant pramlintide precursor.

Pramlintide is an artificially designed protein which has the same function as amylin in human body. This protein is extremely difficult to synthesize through prokaryotic expression method because of its two essential active sites, intrachain disulfide bond and C-terminal amide group. Since α-amidating monooxygenase is widely distributed in human and animal, it is possible to use pramlintide precursor with an additional C-terminal glycine (PAG), which is the potential substrate of α-amidating monooxygenase, for in vivo applications. The recombinant PAG was expressed in Escherichia coli using the small ubiquitin-related modifier (SUMO) as the molecular chaperone, and the optimal fusion expression level reached to 36.3% of the total supernatant protein. Under optimal conditions in a 10-L fermentor, the recombinant PAG was obtained with a purity of greater than 95%, and the average expression level was reached to 20 mg/L. The authenticity and the intrachain disulfide bridge of PAG were confirmed by Western blotting and matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectrometry (MALDI-TOF MS) as well as N-terminal sequencing of protein. Based on an L6 myoblast cell model in vitro and an animal model of gastric emptying in vivo, the results of activity revealed that PAG showed a lower biological activity in vitro but has almost the same activity as the chemically synthesized pramlintide in vivo.

Molecular modeling of Gram-positive bacteria peptidoglycan layer, selected glycopeptide antibiotics and vancomycin derivatives modified with sugar moieties

Proper understanding of the mechanisms of binding to Gram-positive bacteria cell wall layers—especially to the peptidoglycan (PG) layer, seems to be crucial for proper development of new drug candidates which are effective against these bacteria.In this work we have constructed two different models of the Gram-positive bacteria PG layer: the layered and the scaffold models. PG conformational changes during geometry optimization, models relaxation, and molecular dynamics were described and discussed. We have found that the border surface of both PG layer models differs from the surface located away from the edge of models and the chains formed by disaccharide units prefer helix-like conformation. This curling of PG chains significantly affects the shape of antibiotic-accessible surface and the process is thus crucial for new drug development.Glycopeptide antibiotics effective against Gram-positive bacteria, such as vancomycin and its semisynthetic derivatives—oritavancin and telavancin, bind to d-alanyl-d-alanine stem termini on the peptidoglycan precursors of the cell wall. This binding inhibits cross-linking between the peptides and subsequently prevents cell wall synthesis.In this study some of the aspects of conformational freedom of vancomycin and restrictions from the modifications of vancomycin structure introduced into oritavancin and telavancin and five other vancomycin derivatives (with addition of 2-acetamido-2-deoxy-β-d-galactopyranosylamine, 2-acetamido-2-deoxy-β-d-glucopyranosylamine, 1-amine-1-deoxy-d-glucitol, 2-amino-2-deoxy-d-galactitol, or 2-amino-2-deoxy-d-glucitol to the C-terminal amino acid group in the vancomycin) are presented and discussed. The resulting molecular dynamics trajectories, root mean square deviation changes of aglycon and saccharide moieties as well as a comparative study of possible interactions with cyclic and chain forms of modified groups have been carried out, measured, and analyzed. Energetically advantageous conformations show close similarity to the structures known from the experimental data, but the diversity of others suggest very high conformational freedom of all modeled antibiotics and vancomycin derivatives. Alditol derivatives move closer to the peptidoglycan chain more easily but they also form intramolecular interactions more frequently than their homologous cyclic forms. One of the proposed derivatives seems to be a promising agent which is efficient in treatment of infections caused by Gram-positive bacteria.

Toward engineering efficient peptidomimetics. Screening conformational landscape of two modified dehydroaminoacids

Effective peptidomimetics should posses structural rigidity and appropriate interaction pattern leading to potential spatial and electronic matching to the target receptor site. Rational design of such small bioactive molecules could push chemical synthesis and molecular modeling toward faster progress in medicinal chemistry. Conformational properties of N-t-butoxycarbonyl-glycine-(E/Z)-dehydrophenylalanine N',N'-dimethylamides (Boc-Gly-(E/Z)-ΔPhe-NMe2 ) in chloroform were studied by NMR and IR spectroscopy. The experimental findings were supported by extensive calculations at DFT(B3LYP, M06-2X) and MP2 levels of theory and the β-turn tendency for both isomers of the studied dipeptide were determined in vacuum and in solution. The theoretical data and experimental IR results were used as an additional information for the NMR-based determination of the detailed solution conformations of the peptides. The obtained results reveal that N-methylation of C-terminal amide group changes dramatically the conformational properties of studied dehydropeptides. Theoretical conformational analysis reveals that the tendency to adopt β-turn conformations is much weaker for the N-methylated Z isomer (Boc-Gly-(Z)-ΔPhe-NMe2 ), both in vacuum and in polar environment. On the contrary, N-methylated E isomer (Boc-Gly-(E)-ΔPhe-NMe2 ) can easily adopt β-turn conformation, but the backbone torsion angles (φ1, ψ1, φ2, ψ2) are off the limits for common β-turn types.

Selective CGRP and adrenomedullin peptide binding by tethered RAMP‐calcitonin receptor‐like receptor extracellular domain fusion proteins

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are related peptides that are potent vasodilators. The CGRP and AM receptors are heteromeric protein complexes comprised of a shared calcitonin receptor-like receptor (CLR) subunit and a variable receptor activity modifying protein (RAMP) subunit. RAMP1 enables CGRP binding whereas RAMP2 confers AM specificity. How RAMPs determine peptide selectivity is unclear and the receptor stoichiometries are a topic of debate with evidence for 1:1, 2:2, and 2:1 CLR:RAMP stoichiometries. Here, we describe bacterial production of recombinant tethered RAMP-CLR extracellular domain (ECD) fusion proteins and biochemical characterization of their peptide binding properties. Tethering the two ECDs ensures complex stability and enforces defined stoichiometry. The RAMP1-CLR ECD fusion purified as a monomer, whereas the RAMP2-CLR ECD fusion purified as a dimer. Both proteins selectively bound their respective peptides with affinities in the low micromolar range. Truncated CGRP(27-37) and AM(37-52) fragments were identified as the minimal ECD complex binding regions. The CGRP C-terminal amide group contributed to, but was not required for, ECD binding, whereas the AM C-terminal amide group was essential for ECD binding. Alanine-scan experiments identified CGRP residues T30, V32, and F37 and AM residues P43, K46, I47, and Y52 as critical for ECD binding. Our results identify CGRP and AM determinants for receptor ECD complex binding and suggest that the CGRP receptor functions as a 1:1 heterodimer. In contrast, the AM receptor may function as a 2:2 dimer of heterodimers, although our results cannot rule out 2:1 or 1:1 stoichiometries.

Characterization of opioid activities of endomorphin analogs with C-terminal amide to hydrazide conversion

Previously, we have synthesized an endomorphin-2 (EM-2) analog with C-terminal amide to hydrazide conversion, exhibiting slightly lower μ-affinity than EM-2. In the present study, the influence of C-terminal amide group to hydrazide conversion on the in vitro and in vivo opioid activities of EMs was evaluated. Our results demonstrated that C-terminal amide to hydrazide conversion of EMs did not markedly change their μ-opioid receptor binding affinities. Nevertheless, EM-2-NHNH2 decreased guinea pig ileum (GPI) and mouse vas deferens (MVD) potencies by about 10- and 5-fold compared to the parent compound, respectively. It is noteworthy that EM-1-NHNH2 exhibited the highest antinociception after intracerebroventricular (i.c.v.) injection, about 1.5-fold more potent than EM-1, but with moderate colonic contractile and expulsive effects, comparable with EM-1. Additionally, though EM-2-NHNH2 showed a slightly lower antinociceptive effect than EM-2, at higher doses (i.c.v., 1.5 and 5nmol/mouse) the inhibitory effects of colonic propulsion were significantly attenuated, which would be helpful in the development of suitable μ-opioid therapeutics, but without some undesirable side effects. Therefore, the present results gave the evidence that C-terminal amide to hydrazide conversion of EMs may play an important role in the regulation of opioid activities.

N-(L-2-Aminopentanoyl)-L-phenylalanine dihydrate, a hydrophobic dipeptide with a nonproteinogenic residue

The title dipeptide, better known as L-norvalyl-L-phenylalanine {systematic name: (S)-2-[(S)-2-aminopentanamido]-3-phenylpropanoic acid dihydrate}, C14H20N2O3·2H2O, has a nonproteinogenic N-terminal residue. In the solid state, it takes on a molecular conformation typical for one of the three classes of nanoporous dipeptides, but like two related compounds with a hydrophobic N-terminal residue and a C-terminal L-phenylalanine, it fails to form channels or pores. Instead, the crystal structure is divided into distinct hydrophobic and hydrophilic layers, the latter encompassing cocrystallized water molecules connecting the charged N- and C-terminal groups.

Solid phase synthesis of Smac/DIABLO-derived peptides using a ‘Safety-Catch’ resin: Identification of potent XIAP BIR3 antagonists

The N-terminal sequence of the Smac/DIABLO protein is known to be involved in binding to the BIR3 domain of the anti-apoptotic proteins IAPs, antagonizing their action. Short peptides and peptide mimetics based on the first 4-residues of Smac/DIABLO have been demonstrated to re-sensitize resistant cancer cells, over-expressing IAPs, to apoptosis. Based on the well-defined structural basis for this interaction, a small focused library of C-terminal capped Smac/DIABLO-derived peptides was designed in silico using docking to the XIAP BIR3 domain. The top-ranked computational hits were conveniently synthesized employing Solid Phase Synthesis (SPS) on an alkane sulfonamide ‘Safety-Catch’ resin. This novel approach afforded the rapid synthesis of the target peptide library with high flexibility for the introduction of various C-terminal amide-capping groups. The library members were obtained in high yield (>65%) and purity (>85%), upon nucleophilic release from the activated resin by treatment with various amine nucleophiles. In vitro caspase-9 activity reconstitution assays of the peptides in the presence of the recombinant BIR3-domain of human XIAP (500nM) revealed N-methylalanyl-tertiarybutylglycinyl-4-(R)-phenoxyprolyl-N-biphenylmethyl carboxamide (11a) to be the most potent XIAP BIR3 antagonist of the series synthesized inducing 93% recovery of caspase-9 activity, when used at 1μM concentration. Compound (11a) also demonstrated moderate cytotoxicity against the breast cancer cell lines MDA-MB-231 and MCF-7, compared to the Smac/DIABLO-derived wild-type peptide sequences that were totally inactive in the same cell lines.

Ube2W conjugates ubiquitin to α-amino groups of protein N-termini

The covalent attachment of the protein ubiquitin to intracellular proteins by a process known as ubiquitylation regulates almost all major cellular systems, predominantly by regulating protein turnover. Ubiquitylation requires the co-ordinated action of three enzymes termed E1, E2 and E3, and typically results in the formation of an isopeptide bond between the C-terminal carboxy group of ubiquitin and the ϵ-amino group of a target lysine residue. However, ubiquitin is also known to conjugate to the thiol of cysteine residue side chains and the α-amino group of protein N-termini, although the enzymes responsible for discrimination between different chemical groups have not been defined. In the present study, we show that Ube2W (Ubc16) is an E2 ubiquitin-conjugating enzyme with specific protein N-terminal mono-ubiquitylation activity. Ube2W conjugates ubiquitin not only to its own N-terminus, but also to that of the small ubiquitin-like modifier SUMO (small ubiquitin-related modifier) in a manner dependent on the SUMO-targeted ubiquitin ligase RNF4 (RING finger protein 4). Furthermore, N-terminal mono-ubiquitylation of SUMO-2 primes it for poly-ubiquitylation by the Ubc13–UEV1 (ubiquitin-conjugating enzyme E2 variant 1) heterodimer, showing that N-terminal ubiquitylation regulates protein fate. The description in the present study is the first of an E2-conjugating enzyme with N-terminal ubiquitylation activity, and highlights the importance of E2 enzymes in the ultimate outcome of E3-mediated ubiquitylation.

C-terminal methylation of truncated neuropeptides: An enzyme-assisted extraction artifact involving methanol

Neuropeptides are the largest class of signaling molecules used by nervous systems. Today, neuropeptide discovery commonly involves chemical extraction from a tissue source followed by mass spectrometric characterization. Ideally, the extraction procedure accurately preserves the sequence and any inherent modifications of the native peptides. Here, we present data showing that this is not always true. Specifically, we present evidence showing that, in the lobster Homarus americanus, the orcokinin family members, NFDEIDRSGFG-OMe and SSEDMDRLGFG-OMe, are non-native peptides generated from full-length orcokinin precursors as the result of a highly selective peptide modification (peptide truncation with C-terminal methylation) that occurs during extraction. These peptides were observed by MALDI-FTMS and LC–Q-TOFMS analyses when eyestalk ganglia were extracted in a methanolic solvent, but not when tissues were dissected, co-crystallized with matrix, and analyzed directly with methanol excluded from the sample preparation. The identity of NFDEIDRSGFG-OMe was established using MALDI-FTMS/SORI-CID, LC–Q-TOFMS/MS, and comparison with a peptide standard. Extraction substituting deuterated methanol for methanol confirmed that the latter is the source of the C-terminal methyl group, and MS/MS confirmed the C-terminal localization of the added CD3. Surprisingly, NFDEIDRSGFG-OMe is not produced via a chemical acid-catalyzed esterification. Instead, the methylated peptide appears to result from proteolytic truncation in the presence of methanol, as evidenced by a reduction in conversion with the addition of a protease-inhibitor cocktail; heat effectively eliminated the conversion. This unusual and highly specific extraction-derived peptide conversion exemplifies the need to consider both chemical and biochemical processes that may modify the structure of endogenous neuropeptides.

PH-Independence of Trialanine and the Effects of Termini Blocking in Short Peptides: A Combined Vibrational, NMR, UVCD, and Molecular Dynamics Study

Several lines of evidence now well establish that unfolded peptides in general, and alanine in specific, have an intrinsic preference for the polyproline II (pPII) conformation. Investigation of local order in the unfolded state is, however, complicated by experimental limitations and the inherent dynamics of the system, which has in some cases yielded inconsistent results from different types of experiments. One method of studying these systems is the use of short model peptides, and specifically short alanine peptides, known for predominantly sampling pPII structure in aqueous solution. Recently, He et al. ( J. Am. Chem. Soc. 2012 , 134 , 1571 - 1576 ) proposed that unblocked tripeptides may not be suitable models for studying conformational propensities in unfolded peptides due to the presence of end effect, that is, electrostatic interactions between investigated amino acid residues and terminal charges. To determine whether changing the protonation states of the N- and C-termini influence the conformational manifold of the central amino acid residue in tripeptides, we have examined the pH-dependence of unblocked trialanine and the conformational preferences of alanine in the alanine dipeptide. To this end, we measured and globally analyzed amide I' band profiles and NMR J-coupling constants. We described conformational distributions as the superposition of two-dimensional Gaussian distributions assignable to specific subspaces of the Ramachandran plot. Results show that the conformational ensemble of trialanine as a whole, and the pPII content (χpPII = 0.84) in particular, remains practically unaffected by changing the protonation state. We found that compared to trialanine, the alanine dipeptide has slightly lower pPII content (χpPII = 0.74) and an ensemble more reminiscent of the unblocked Gly-Ala-Gly model peptide. In addition, a two-state thermodynamic analysis of the conformational sensitive Δε(T) and (3)J(H(N)H(α))(T) data obtained from electronic circular dichroism and H NMR spectra indicate that the free energy landscape of trialanine is similar in all protonation states. MD simulations for the investigated peptides corroborate this notion and show further that the hydration shell around unblocked trialanine is unaffected by the protonation/deprotonation of the C-terminal group. In contrast, the alanine dipeptide shows a reduced water density around the central residue as well as a less ordered hydration shell, which decreases the pPII propensity and reduces the lifetime of sampled conformations.

Effect of Protonation State on the Stability of Amyloid Oligomers Assembled from TTR(105-115).

Amyloid fibrils are self-assembled aggregates of polypeptides that are implicated in the development of several human diseases. A peptide derived from amino acids 105-115 of the human plasma protein transthyretin forms homogeneous and well-defined fibrils and, as a model system, has been the focus of a number of studies investigating the formation and structure of this class of aggregates. Self-assembly of TTR(105-115) occurs at low pH, and this work explores the effect of protonation on the growth and stability of small cross-β aggregates. Using molecular dynamics simulations of structures up to the decamer in both protonated and deprotonated states, we find that, whereas hexamers are more stable for protonated peptides, higher order oligomers are more stable when the peptides are deprotonated. Our findings imply a change in the acid pK of the protonated C-terminal group during the formation of fibrils, which leads to stabilization of higher-order oligomers through electrostatic interactions.

Total synthesis of biotinylated N domain of human hepatocyte growth factor

Hepatocyte growth factor/scatter factor (HGF/SF) is the high affinity ligand of MET tyrosine kinase receptor. We report here the total synthesis of a biotinylated analogue of human HGF/SF N domain. Functionally, N domain is part of the HGF/SF high affinity binding site for MET and also the main HGF/SF binding site for heparin. The 97 Aa linear chain featuring a C-terminal biotin group was assembled in high yield using an N-to-C one-pot three segments assembly strategy relying on a sequential Native Chemical Ligation (NCL)/bis(2-sulfanylethyl)amido (SEA) native peptide ligation process. The folded protein displayed the native disulfide bond pattern and showed the ability to bind heparin.

Three-dimensional structure of carboxypeptidase T from Thermoactinomyces vulgaris in complex with N-BOC-L-leucine

The 3D structure of recombinant bacterial carboxypeptidase T (CPT) in complex with N-BOC-L-leucine was determined at 1.38 Å resolution. Crystals for the X-ray study were grown in microgravity using the counter-diffusion technique. N-BOC-L-leucine and SO4(2-) ion bound in the enzyme active site were localized in the electron density map. Location of the leucine side chain in CPT-N-BOC-L-leucine complex allowed identification of the S1 subsite of the enzyme, and its structure was determined. Superposition of the structures of CPT-N-BOC-L-leucine complex and complexes of pancreatic carboxypeptidases A and B with substrate and inhibitors was carried out, and similarity of the S1 subsites in these three carboxypeptidases was revealed. It was found that SO4(2-) ion occupies the same position in the S1' subsite as the C-terminal carboxy group of the substrate.

The Sulfur Carrier Protein TusA Has a Pleiotropic Role in Escherichia coli That Also Affects Molybdenum Cofactor Biosynthesis*

The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes.

Evolution of an amino acid based prodrug approach: stay tuned.

Certain acyclic nucleoside phosphonates (ANPs) such as (S)-HPMPC (cidofovir, Vistide) and (S)-HPMPA have been shown to be active against a broad spectrum of DNA and retroviruses. However, their poor absorption as well as their toxicity limit the utilization of these therapeutics in the clinic. Nucleoside phosphonates are poorly absorbed primarily due to the presence of the phosphonic acid group, which ionizes at physiological pH. When dosed intravenously they display dose-limiting nephrotoxicity due to their accumulation in the kidney. To overcome these limitations, nucleoside phosphonate prodrug strategies have taken center stage in the development pathway and a number of different approaches are at various stages of development. Our efforts have focused on the development of ANP prodrugs in which a benign amino acid promoiety masks a phosphonate P-OH via a hydroxyl side chain. The design of these prodrugs incorporates multiple chemical groups (the P-X-C linkage, the amino acid stereochemistry, the C-terminal and N-terminal functional groups) that can be tuned to modify absorption, pharmacokinetic and efficacy properties with the goal of improving overall prodrug performance.

Fluorescence Anisotropy in a Protein: DNA System Undergoing Inducible Assembly

Fluorescence anisotropy may be used as an indicator of homo-FRET making it an attractive reported of oligomer formation and aggregation. A model system was created consisting of a monomeric teal fluorescent protein peptide nucleic acid (mTFP-PNA) fusion. The mTFP-PNA monomer units produced by expressed protein ligation (EPL). An mTFP with a C-terminal thioester group was expressed with a modified intein and subsequently conjugated to a PNA with an N-terminal thiol group. This generates a native peptide bond at the ligation site. Addition of a complementary template DNA to solutions of mTFP-PNA monomer units resulted in template directed induced assembly. Assembly was confirmed by SDS-PAGE, mass spectrometry, and size exclusion HPLC.Fluorescence anisotropy was monitored over the course of a mTFP-PNA:DNA titration. Template coding for dimer formation was studied. The anisotropy showed a decreasing trend related to homo-FRET in the assembled forms. A maximal reduction (41%) was observed at a DNA to mTFP-PNA ratio of 1:2. Anisotropy, then increased steadily up to a 1:1 ratio.This study demonstrates an inducibly assembled homo-FRET system using expressed protein ligation which may be extended to study oligomerization and cluster formation in living systems.